The Senior Investigator moved to the NCI during FY2011 and hired personnel to set up a research laboratory during the first half of FY2012. A GS-11 Geneticist was recruited in November 2011, initially to set up the laboratory; this scientist then began working on this RMS gene fusion project in March 2012. One postdoctoral trainee was recruited in March 2012 and contributed 20% effort to this RMS gene fusion project. In addition, a second postdoctoral trainee was recruited in July 2012 and will contribute 100% effort to the RMS gene fusion project. As he is just starting in the laboratory at the time of this report, his efforts are not be reflected in this annual report.In our recently published study, fluorescence in situ hybridization analysis of RMS cases with FOXO1 probes demonstrated that 93% of PAX7-FOXO1-expressing and 9% of PAX3-FOXO1-expressing RMS tumors have fusion gene amplification. After developing quantitative RT-PCR methodology that permitted quantification of PAX3-FOXO1 and PAX7-FOXO1 fusion transcripts on a common scale, analysis of a large number of fusion-positive tumors indicated that PAX7-FOXO1-expressing tumors expressed higher levels of fusion transcript than PAX3-FOXO1-expressing tumors (with a 3.7-fold higher mean level). These findings indicate that fusion gene amplification is a more robust mechanism than copy-number independent transcriptional activation for increasing steady state fusion transcript abundance. We wished to further analyze this difference in fusion transcript expression between the two fusion subsets to determine if it leads to a difference in expression of the fusion protein and downstream target genes. There are relatively few tumor specimens in which there is sufficient material for western blot analysis of protein expression. Therefore, in collaboration with Dr. Steve Master at the University of Pennsylvania, we first assayed a number of downstream target genes for expression in fusion-positive tumors. Four genes with known PAX3/PAX7-FOXO1 binding sites were assayed in a series of PAX3-FOXO1- and PAX7-FOXO1-expressing cases. For three of the genes (MYCN, FGFR4, and DAPK1), the PAX7-FOXO1 subset expressed lower or similar levels of target gene expression. For the final target gene examined, RYR3 had 84% higher mean expression level in the PAX7-FOXO1 subset. These findings do not indicate a program of target gene activation corresponding to the increased expression of the fusion gene in the PAX7-FOXO1 subset. To examine the differences between PAX3-FOXO1- and PAX7-FOXO1-expressing tumors in a controlled environment, we collected cell lines from these tumors. In particular, we now have eight PAX3-FOXO1-expressing and two PAX7-FOXO1 expressing RMS cell lines. In addition, we have eight fusion-negative RMS cell lines (including two pleomorphic RMS lines). For western blot studies, we obtained a monoclonal antibody to the C-terminus of FOXO1 and successfully used this antibody to detect wild-type FOXO1 and/or the fusion protein in these RMS cell lines. Studies are in progress to quantify expression of the fusion protein and compare fusion protein expression to fusion transcript expression levels in the fusion-positive cell lines. As part of our studies to identify cis-acting regulatory elements in the 3' region of the FOXO1 gene, we analyzed the available genomic information on this region on the UCSC Genome Browser. We reasoned that this hypothetical 3' FOXO1 element should be 3' of all rearrangements involving the first intron of FOXO1 and 5' of the next gene on chromosome 13. Based on the available mapping information, the potential localization for this 3' FOXO1 element is present within an 80 kb region. From CHiP seq data for the H3K27Ac mark in 7 cell lines from ENCODE, there are three strong clusters of sites for this chromatin mark, which is known to be found near active regulatory elements. One site was localized in the vicinity of the last two exons of FOXO1 and the other two sites were in the intergenic region between FOXO1 and the adjacent TTL gene. These three clusters coincided with clusters of digital DNase I hypersensitivity sites from ENCODE and with clusters of transcription factor binding sites as determined by ChIP-Seq studies from ENCODE. To generate a reporter system to detect 3' FOXO1 fragments that synergize with the 5' PAX3 promoter, we cloned a 2 kb 5' PAX3 fragment in front of the firefly luciferase reporter cDNA in the PGL3 reporter construct (Promega). This construct has cloning sites downstream of the polyA signal to screen for 3' enhancers. Two approaches are being used to identify the postulated cis-acting 3' FOXO1 regulatory elements. In the first approach (direct approach), we used PCR to isolate the three regions with H3K27Ac marks described above. As these DNA regions measure up to 5-10 kb, primers were developed to amplify a series of overlapping 1 kb fragments. In all, 22 fragments from the three clusters were amplified and subcloned into the reporter constructs. As a second approach (shotgun approach), a 160 kb BAC clone that covers the 80 kb intergenic region (as well as portions of the FOXO1 and TTL genes) has been cut by partial digestion with two frequent 4-base pair recognizing restriction enzymes and then fragments measuring 1 kb in size will be isolated and cloned into the 5' PAX3 luciferase reporter plasmid. For each approach, the various subclones of the reporter construct and a control constitutive renilla luciferase reporter will be transfected into a myogenic cell line, and the reporter activity measured to determine which inserts have enhancing activity.